During the first reporting period, the project established all major experimental systems required to address the aims outlined in the Description of the Action. These platforms—enabling rRNA-modification mapping and functional analysis of ribosome heterogeneity—are now fully operational and routinely applied. The work completed to date provides substantial insight into how rRNA modifications influence lineage decisions and cellular identity across the hematopoietic hierarchy.
A central achievement is the comprehensive mapping of rRNA modifications across multiple hematopoietic stem and progenitor populations. Using optimized purification strategies, we isolated well-defined subsets—including HSCs, CMPs, GMPs, MEPs, CLPs, and committed erythroid progenitors (preMegE, preCFUE, CFUE), as well as B-lymphocyte progenitors—and profiled both 2’-O-methylation (2’OMe) and pseudouridine (Ψ). This analysis revealed striking lineage-specific patterns: myeloid cells display highly dynamic 2’OMe levels with stable Ψ, whereas lymphoid progenitors show dynamic Ψ changes with relatively stable 2’OMe. These findings constitute the first systematic demonstration of cell-type-specific rRNA modification landscapes in hematopoiesis.
We next assessed the functional consequences of individual modification sites. Using targeted perturbations, we identified three 2’OMe sites whose loss affects proliferation, differentiation, or both, demonstrating that rRNA modifications exert discrete regulatory functions. Among these, Gm4588 emerged as a key site. Ex-vivo studies showed that Gm4588 loss compromises HSC self-renewal, establishing a causal link between rRNA methylation and stem-cell maintenance.
To evaluate in-vivo relevance, CRISPR-edited HSCs lacking Gm4588 were transplanted into lethally irradiated mice. Although capable of reconstitution, Gm4588-KO cells exhibited markedly reduced fitness, reduced bone-marrow contribution, and impaired proliferation. In competitive transplantations, they were rapidly outcompeted and became nearly undetectable, confirming that Gm4588 is essential for stem-cell fitness in vivo.
Further in-vivo analysis revealed that secondary transplant recipients of Gm4588-KO HSCs developed pre-leukemic features, including expansion of myeloid progenitors at the expense of lymphoid and erythroid lineages. This phenotype suggests a possible connection between disrupted rRNA methylation and malignant evolution. Tertiary transplantations are planned to test progression toward leukemia.
Finally, we successfully implemented ultra-low-input ribosome profiling in bone-marrow erythroid populations. This enabled quantification of translation across defined stages—from HSCs to committed CFUE cells—and revealed dynamic, stage-specific translational programs. These results establish translational output as a sensitive indicator of lineage identity and differentiation state.
